Glucose-6-phosphatase (G6Pase; EC 3.1.3.9) catalyzes hydrolysis of glucose-6-phosphate (G6P), yielding glucose. This reaction is the terminal step in the gluconeogenic and glycogenolytic pathways.
Most cells of the body are able to convert glucose absorbed from the blood stream to G6P, thereby preventing facilitated diffusion of the glucose moiety out of the cell. Some cells, such as liver cells, possess G6Pase activity, whereby G6P can be converted to glucose and released to the bloodstream or used by the cell for metabolism. For example, formation of glucose in the liver from hepatically-stored glycogen (i.e., involving intermediate hydrolysis of G6P by G6Pase) is an important mechanism by which blood glucose is maintained at a normal level between meals.
Maintenance of normal blood glucose levels is important for nutrition of certain tissues (e.g., brain and other nervous system tissues and gonadal germinal epithelium) which are substantially incapable of metabolizing other energy sources such as fatty acids or amino acids. Lipid and protein metabolism can be undesirable, in that such metabolism depletes bodily stores of lipids and proteins, and in that the by-products of such metabolism (e.g., certain lipoprotein-containing particles) can cause or contribute to pathological conditions (e.g., deposition of lipoprotein plaque in arteries). Thus, in addition to providing nutrition to tissues which metabolize glucose almost exclusively, maintenance of normal blood glucose levels prevents physiologically inappropriate reliance of the body on non-carbohydrate catabolic routes.
In diabetic patients, in whom aberrantly diminished secretion of insulin leads to defects in carbohydrate metabolism, fat metabolism is abnormally increased, leading to greater-than-normal levels of circulating fatty acids, which in turn cause greater-than-normal deposition of cholesterol and other plaque materials in arteries. Indeed, abnormalities in fat and protein metabolism are common in diabetics, and account for much of the morbidity and mortality experienced by such patients, including acidosis, arteriosclerosis, coronary artery disease and other circulatory disorders, and wasting disease conditions (i.e., attributable to aberrant protein degradation).
In normal patients, blood insulin level during fasting is relatively constant, but increases in a two-stage manner upon influx of glucose, certain amino acids (e.g., lysine, arginine, and alanine), or particularly both, into the blood stream. A rapid increase in insulin, attributable to release of pre-formed insulin stored in secretory granules of pancreas islet of Langerhans beta cells occurs in the first stage, followed by more gradual and pronounced release of presumably newly-synthesized insulin in a second stage. Secretion of glucagon, a hormone secreted by alpha cells of pancreas islet of Langerhans, is also closely regulated in coordination with blood levels of glucose and amino acids.
Although it is known that secretion of insulin and secretion of glucagon are tightly regulated, and that modulation of secretion of these molecules occurs rapidly, the mechanisms by which such secretions are modulated are not fully understood. More particularly, the mechanism by which blood glucose level, blood amino acid levels, or both, affect production, processing, and release of hormones like insulin and glucagon has not been fully elucidated. Further knowledge of the physiological mechanisms by which these processes are regulated would enable medical practitioners to more predictably and efficaciously prognosticate, diagnose, inhibit, prevent, alleviate, or even cure both hormone-associated metabolic disorders (e.g., diabetes and hyperinsulinism) and undesirable physiological phenomena (e.g. atherosclerosis, tissue wasting) that accompany such disorders.
Previously characterized G6Pase enzymes isolated from liver and kidney tissues are believed to be localized at the membrane of the endoplasmic reticulum (Ebert et al., 1999, Diabetes 48:543-554; Burchell, 1990, FASEB J. 4:2978-2988; Mithieux, 1997, Eur. J. Endocrinol. 136:137-145; Foster et al., 1997, Proc. Soc. Exp. Biol. Med. 215:314-332) and are also believed to be associated with one or more proteins which facilitate transmembrane transport of glucose-6-phostphate, glucose, or both (Gerin et al., 1997, FEBS Lett. 419:235-238; Waddell et al., 1992, Biochem. J. 286:173-177). Genes encoding G6Pase enzymes and catalytic subunits thereof have been cloned in humans and mice (Lei et al., 1993, Science 262:580-583; Shelly et al., 1993, J. Biol. Chem. 268:21482-21485). Pancreatic G6Pase is distinct from the hepatic and kidney forms of this enzyme, and appears to be present in the endoplasmic reticulum of murine pancreatic islet of Langerhans cells of the alpha and beta types, likely in the form of a multi-protein complex. (Arden et al., 1999, Diabetes 48:531-542; Trinh et al., 1997, J. Biol. Chem. 272:24837-24842).
Human pancreatic G6Pase has not previously been isolate, nor has its sequence been determined. A need remains for isolation and sequencing of the human gene encoding the catalytic subunit of human pancreatic islet cell-specific G6Pase. The present invention satisfies this need.
The present invention is based, at least in part, on the discovery of a cDNA molecule encoding the catalytic subunit of human pancreatic islet cell-specific G6Pase. This protein and fragments, derivatives, and variants thereof are collectively referred to as polypeptides of the invention or proteins of the invention. Nucleic acid molecules encoding polypeptides of the invention are among those collectively referred to as nucleic acid molecules of the invention.
Polypeptides of the invention include the catalytic subunit of human pancreatic islet cell-specific G6Pase (xe2x80x9ch-ig6pxe2x80x9d) and proteins which exhibit significant homology therewith (i.e., proteins having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99% or more identical to SEQ ID NO: 3). Other polypeptides of the invention include those which comprise (or consist of) a biologically active portion of h-ig6p (e.g., a portion which exhibits a catalytic activity of h-ig6p), a structural feature (e.g., an epitope or secondary structural domain) of h-ig6p, a functional portion of h-ig6p (e.g., a portion which binds a physiological substrate), or some combination of these.
Nucleic acid molecules of the invention include those which encode any of the polypeptides of the invention (e.g., a nucleic acid molecule that encodes the entire catalytic subunit of human pancreatic islet cell-specific G6Pase). By way of example, such nuclein acid molecules can have a nucleotide sequence that comprises (or consists of) all, or a portion, of one of SEQ ID NO: 1, SEQ ID NO: 2, and the nucleotide sequence of the clone deposited with American Type Culture Collection (ATCC) on Jul. 28, 2000 as accession number PTA-2282, or a complement of one of these sequences. Nucleic acids of the invention can, alternatively, have a nucleotide sequence that is at least 91% (or 92%, 95%, 98%, or 99% or more) identical to one of these sequences, particularly where the sequence identity is such that some or all of the amino acid residues described herein as having structural, functional, or catalytic relevance are preserved.
The invention also includes nucleic acid molecules which do not necessarily encode a polypeptide of the invention, but which are nonetheless suitable, for example, as a hybridization probe for detection of a nucleic acid encoding a polypeptide of the invention or as a primer for amplifying (or replicating) all or a portion of such a nucleic acid.
Also included within the scope of the invention are modulators of polypeptides and nucleic acid molecules of the invention and methods for making and identifying such modulators. Examples of such modulators include antibodies which bind specifically with h-ig6p (i.e., with an epitope of h-ig6p) and h-ig6p-binding fragments of such antibodies. Other examples of modulators of the invention include anti-sense nucleic acid molecules which are capable of hybridizing with a nucleic acid molecule of the invention (particularly including those which hybridize under stringent binding conditions) and inhibiting (or even preventing) expression thereof.
The nucleic acid molecules, polypeptides, and modulators of the invention are useful as modulating agents for regulating a variety of cellular processes, particularly including cellular processes which occur in human pancreatic islet of Langerhans cells (e.g., in alpha cells, in beta cells, or both), or in testis cells. Examples of these cellular processes include interaction of blood glucose with alpha and beta cells and subcellar components thereof, secretion of insulin by beta cells, secretion of glucagon by alpha cells, secretion of testosterone (or another testis-derived hormone) from testis cells, and transmembrane transport of glucose (optionally coupled with phosphorylation of glucose) or of G6P (optionally coupled with de-phosphorylation of G6P) by alpha or beta cells of the pancreas or by testis cells. The membrane across which the hormone is transported can be, for example, the cytoplasmic membrane or the membrane surrounding the endoplasmic reticulum. These cellular processes are involved in homeostasis in humans not afflicted with a pancreatic disorder, and can also be involved in development or manifestation of a pancreatic disease state. These cellular processes are also involved in normal and aberrant secretion of testosterone (and other testis-derived hormones) from testes in men.
The invention thus includes methods of inhibiting, preventing, prognosticating, diagnosing, or treating disorders which are associated with aberrant expression or activity of h-ig6p, including pancreatic and testicular disorders. Examples of such disorders include diabetes (e.g., type 2 diabetes, maturity-onset diabetes of the young, and the like), hyperinsulinism, glycogen storage diseases, hypogonadism, baldness, male developmental disorders (e.g., Klinefelter""s syndrome, anorchia, and Noonan""s syndrome), male infertility, androgeny, pseudohermaphroditism, and hermaphroditism. Pharmaceutical compositions comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a modulator of the invention, and combinations of these are included within the scope of the invention.
Other features and advantages of the invention will be apparent from the following detailed description and claims.